Drug composition comprising dipeptidyl aldehyde derivative

ABSTRACT

The present invention provides a preparation containing a compound of the formula (I)  
                 
 
     wherein R 1  is an alkyl group having 1 to 4 carbon atoms or an optionally substituted aryl group having 6 to 10 carbon atoms; R 2  and R 3  are the same or different and each is hydrogen or an alkyl group having 1 to 4 carbon atoms, or R 2  and R 3  in combination form a ring having 3 to 7 carbon atoms; and R 4  is a lower alkyl group optionally substituted by aryl, cycloalkyl or aromatic heterocyclic residue, or a pharmaceutically acceptable salt thereof together with a lipid. The preparation shows improved stability and improved absorption and penetration into tissue.

TECHNICAL FIELD

[0001] The present invention relates to a pharmaceutical compositioncomprising a dipeptidyl aldehyde derivative and a lipid. In addition,the present invention relates to a method for improving the stability ofa dipeptidyl aldehyde derivative and a method for promoting absorptionor improving penetration into tissue.

BACKGROUND ART

[0002] A compound of the formula (I)

[0003] wherein R¹ is an alkyl group having 1 to 4 carbon atoms or anoptionally substituted aryl group having 6 to 10 carbon atoms; R² and R³are the same or different and each is a hydrogen or an alkyl grouphaving 1 to 4 carbon atoms, or R² and R³ in combination form a ringhaving 3 to 7 carbon atoms; and R⁴ is a lower alkyl group optionallysubstituted by aryl, cycloalkyl or aromatic heterocyclic residue[hereinafter sometimes to be referred to as dipeptidyl aldehydederivative (I)] and a pharmaceutically acceptable salt thereof aredisclosed in JP-A-10-147564 (U.S. Pat. No. 6,214,800). This compound hasan inhibitory activity on cysteine protease such as calpain and thelike, and is known to be useful as a pharmaceutical preparation for theprophylaxis or treatment of cysteine protease-associated diseases, suchas ischemic diseases, immune diseases, Alzheimer's disease,osteoporosis, diseases caused by brain tissue impairment (e.g., cerebralvasospasm, cerebral thrombosis, cerebral infarction, cerebral embolism,intracerebral hemorrhage, subarachnoid hemorrhage, hypertensiveencephalopathy, transient cerebral ischemic attack, multiinfarctdementia, cerebral arteriosclerosis, Huntington's disease and the like),cataract, glaucoma (e.g., open angle glaucoma, low tension glaucoma,closed angle glaucoma and the like), retinochoroidal disorders (e.g.,retinal vascular abnormalities such as occlusion of retinal vessels,retinal periphlebitis, Eales' disease, ischemic ophthalmic syndrome,retinal arteriolar macroaneurysm and the like, retinopathy due tohypertension or renal disease, diabetic retinopathy, retinal pigmentepitheliopathy, retinal dystrophy, macular dystrophy, retinochoroidalatrophy, chorioretinopathy, macular degeneration, macular edema,detachment of the retinal pigment epithelium, detachment of the retina,degenerative retinoschisis, retinoblastoma, retinal pigment epitheliumtumor, optic disc capirally angioma and the like), eyeball posteriorcomplications due to photocoagulation (e.g., macular edema, detachmentof the retina, optic neuritis, visual field abnormalities, disturbanceof light sense, color vision deficiency and the like) and the like, oras an agent for the prophylaxis or treatment of angiogenesis, detachmentof the retina and the like [JP-A-10-147566 (U.S. Pat. No. 6,214,800),WO99/48522, WO99/44626].

[0004] However, dipeptidyl aldehyde derivative (I) is hardly soluble inwater and when dissolved using a solubilizer, it becomes unstable. Whenused for oral administration or topical administration, moreover,absorption and penetration into tissue of this derivative afteradministration are not satisfactory. For sufficient exhibition ofefficacy, therefore, the dose needs to be increased.

[0005] As a pharmaceutical preparation containing a lipid, liposome isknown. Liposome can be used for the targeting of a drug, because it issuperior in compatibility with living organisms, permits easy control ofsurface properties, size and the like, permits enclosure of variouscompounds having diverse properties, and the like [D. D. Lasic,“Liposomes: from basic to applications”, Elsevier Science Publishers,pp. 261-471 (1993)]. In practice, however, liposome is associated withproblems in that the inclusion rate of drug into liposome isimpractically low and the like, due to which it has so far produced fewexamples of clinical success, such as a liposome preparation ofdoxorubicin and the like [D. D. Lasic, Nature 380, 561-562 (1996)].

DISCLOSURE OF THE INVENTION

[0006] The invention aims at creation of a pharmaceutical composition ofdipeptidyl aldehyde derivative (I) that shows fine absorption by oraladministration, and a pharmaceutical composition that enhancespenetration thereof into tissue by topical administration. Further, itaims at creation of a pharmaceutical composition of dipeptidyl aldehydederivative (I), which is stable over a long period of time even in waterand which can be administered by injection or instillation into the eye.

[0007] Accordingly, the present invention relates to (1) apharmaceutical composition containing a compound represented by theformula (I)

[0008] wherein

[0009] R¹ is an alkyl group having 1 to 4 carbon atoms or an optionallysubstituted aryl group having 6 to 10 carbon atoms;

[0010] R² and R³

[0011] are the same or different and each is hydrogen or an alkyl grouphaving 1 to 4 carbon atoms, or R² and R³ in combination form a ringhaving 3 to 7 carbon atoms; and

[0012] R⁴ is a lower alkyl group optionally substituted by aryl,cycloalkyl or aromatic heterocyclic residue,

[0013] or a pharmaceutically acceptable salt thereof and a lipid,

[0014] (2) the pharmaceutical composition of the above-mentioned (1),wherein R¹ in the formula (I) is phenyl or naphthyl, which is optionallysubstituted by fluorine, chlorine or methyl,

[0015] (3) the pharmaceutical composition of the above-mentioned (1),wherein R¹ in the formula (I) is a group selected from methyl,4-fluorophenyl, 4-chlorophenyl, p-tolyl and 2-naphthyl,

[0016] (4) the pharmaceutical composition of the above-mentioned (1),wherein, in the formula (I), R² is propyl, isopropyl or tert-butyl, andR³ is hydrogen,

[0017] (5) the pharmaceutical composition of the above-mentioned (1),wherein, in the formula (I), R² is isopropyl and R³ is hydrogen,

[0018] (6) the pharmaceutical composition of the above-mentioned (1),wherein, in the formula (I), R² and R³ in combination formcyclopentylidene or cyclohexylidene,

[0019] (7) the pharmaceutical composition of the above-mentioned (1)wherein, in the formula (I), R⁴ is a group selected from isobutyl,benzyl, cyclohexylmethyl and indol-3-yl-methyl,

[0020] (8) the pharmaceutical composition of the above-mentioned (1),wherein the compound of the formula (I) isN-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal or a pharmaceuticallyacceptable salt thereof,

[0021] (9) the pharmaceutical composition of any of the above-mentioned(1)-(8), wherein a lower limit of the amount of the lipid constitutingthe pharmaceutical composition is about 5 parts by weight and an upperlimit thereof is about 14 parts by weight, per 1 part by weight of thecompound of the formula (I) or a pharmaceutically acceptable saltthereof,

[0022] (10) the pharmaceutical composition of the above-mentioned (9),wherein the compound of the formula (I) or a pharmaceutically acceptablesalt thereof and a lipid form a complex,

[0023] (11) the pharmaceutical composition of the above-mentioned (10),wherein the complex is a liposome,

[0024] (12) the pharmaceutical composition of the above-mentioned (11),wherein the amount of the lipid constituting the liposome is about7-about 14 parts by weight, per 1 part by weight of the compound of theformula (I) or a pharmaceutically acceptable salt thereof,

[0025] (13) the pharmaceutical composition of any of the above-mentioned(1)-(12), which is used for the prophylaxis or treatment of ischemicdisease, immune disease, Alzheimer's disease, osteoporosis, diseases dueto brain tissue impairment, cataract, glaucoma, retinochoroidaldisorder, eyeball posterior complication due to photocoagulation,diseases accompanying angiogenesis and the like,

[0026] (14) the pharmaceutical composition of any of the above-mentioned(1)-(13), which is for an oral administration,

[0027] (15) the pharmaceutical composition of any of the above-mentioned(1)-(13), which is for eye drops,

[0028] (16) the pharmaceutical composition of any of the above-mentioned(1)-(13), which is for an injection,

[0029] (17) a method for improving the stability of a compoundrepresented by the formula (I), which comprises bringing a compoundrepresented by the formula (I) or a pharmaceutically acceptable saltthereof into contact with a lipid,

[0030] (18) a method for promoting the absorption of the compoundrepresented by the formula (I), which comprises bringing the compoundrepresented by the formula (I) or a pharmaceutically acceptable saltthereof into contact with a lipid, and

[0031] (19) a method for improving penetration of the compoundrepresented by the formula (I) into a tissue, which comprises bringingthe compound represented by the formula (I) or a pharmaceuticallyacceptable salt thereof into contact with a lipid.

[0032] In the above-mentioned formula (I), the alkyl group having 1 to 4carbon atoms mentioned for R¹ includes methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl and the like. Of these, methyl ispreferred. The aryl group having 6 to 10 carbon atoms for R¹ includesphenyl, naphthyl, indenyl, azulenyl and the like. Preferred are phenyland naphthyl. The substituent group which may be present on the arylgroup includes, for example, halogen atom (e.g., fluorine, chlorine andthe like), alkyl having 1 to 5 carbon atoms, trifluoromethyl, alkoxyhaving 1 to 5 carbon atoms, hydroxy, acyloxy having 2 to 5 carbon atoms,carboxyl and acyl group having 2 to 5 carbon atoms. Preferred arehalogen atom and alkyl group having 1 to 5 carbon atoms. The morepreferred are fluorine, chlorine and methyl. Preferred examples of theoptionally substituted aryl group having 6 to 10 carbon atoms for R¹ are4-fluorophenyl, 4-chlorophenyl, p-tolyl and 2-naphthyl.

[0033] The alkyl group having 1 to 4 carbon atoms mentioned for R2 andR³ includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl and the like. Preferred are propyl, isopropyl andtert-butyl. The more preferred is isopropyl. Referring to R² and R³, oneof R² and R³ is preferably hydrogen and the other is propyl, isopropyl,isobutyl or tert-butyl. More preferably, R² is propyl, isopropyl,isobutyl or tert-butyl and R³ is hydrogen. Still more preferably, R² isisopropyl and R³ is hydrogen.

[0034] The ring having 3 to 7 carbon atoms which may be formed by R² andR³ in combination includes cyclopropylidene, cyclobutylidene,cyclopentylidene, cyclohexylidene, cycloheptylidene and the like.Cyclopentylidene and cyclohexylidene are particularly preferred.

[0035] The lower alkyl group mentioned for R⁴ includes linear orbranched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, tert-pentyl, hexyl, 4-methylpentyl,1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyland the like. Preferred are methyl and isobutyl. Examples of aryl groupthat optionally substitutes the lower alkyl group for R⁴ include phenyl,1-naphthyl, 2-naphthyl and the like. Of these, phenyl is preferable.Examples of cycloalkyl group that optionally substitutes the lower alkylgroup for R⁴ include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyland the like. Of these, cyclohexyl is preferable. Examples of aromaticheterocyclic residue that optionally substitutes the lower alkyl groupfor R⁴ include monocyclic heterocyclic residue and fused heterocyclicresidue substituted by oxygen, nitrogen and sulfur atom. Examples ofmonocyclic heterocyclic residue include pyrrolyl, furanyl, thiophenyl,oxazolyl, thiazolyl, imidazolyl, pyrazolyl, pyridyl and the like andexamples of fused heterocyclic residue include indolyl, quinolyl,benzothiophenyl, benzofuranyl, indazolyl, quinazolinyl, phthalazinyl,quinoxalinyl and the like. Of these, indolyl is preferable. Preferredexamples of the lower alkyl group optionally substituted by aryl,cycloalkyl or aromatic heterocyclic residue as expressed by R⁴ areisobutyl, benzyl, cyclohexylmethyl and indol-3-ylmethyl.

[0036] The salt of the compound represented by the formula (I) in thepresent invention is preferably a physiologically acceptable salt, whichis exemplified by a salt with an inorganic base, a salt with an organicbase, a salt with an inorganic acid, a salt with an organic acid, a saltwith a basic or an acidic amino acid and the like. Preferable examplesof the salt with inorganic base include alkali metal salts such assodium salt, potassium salt and the like; alkaline earth metal saltssuch as calcium salt, magnesium salt and the like; aluminum salt,ammonium salt and the like. Preferable examples of the salt with organicbase include salts with trimethylamine, pyridine, picoline,ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,N,N-dibenzylethylenediamine and the like. Preferable examples of thesalt with inorganic acid include salts with hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and thelike. Preferable examples of the salt with organic acid include saltswith formic acid, acetic acid, trifluoroacetic acid, fumaric acid,oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Preferable examples of the saltwith basic amino acid include salts with arginine, lysine, ornithine andthe like, and preferable examples of the salt with acidic amino acidinclude salts with aspartic acid, glutamic acid and the like.

[0037] Specific examples of dipeptidyl aldehyde derivative (I) includeN-(2-naphthalenesulfonyl)-L-valyl-L-leucinal,N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal,N-(4-chlorophenylsulfonyl)-L-valyl-L-leucinal,N-(4-methylphenylsulfonyl)-L-valyl-L-leucinal,N-(2-naphthalenesulfonyl)-L-tert-leucyl-L-leucinal,N-(4-fluorophenylsulfonyl)-D-valyl-L-leucinal,N-(4-fluorophenylsulfonyl)-L-norleucyl-L-leucinal,N-(4-fluorophenylsulfonyl)-L-norvalyl-L-leucinal,1-(2-naphthalenesulfonylamino)cyclopentanecarbonyl-L-leucinal,N-(2-naphthalenesulfonyl)-L-tert-leucyl-L-phenylalaninal,N-(4-fluorophenylsulfonyl)-L-valyl-L-phenylalaninal,N-(2-naphthalenesulfonyl)-L-valyl-L-phenylalaninal,N-(4-chlorophenylsulfonyl)-L-valyl-L-phenylalaninal,N-(4-methylphenylsulfonyl)-L-valyl-L-phenylalaninal,N-(2-naphthalenesulfonylamino)cyclohexanecarbonyl-L-phenylalaninal,1-(2-naphthalenesulfonylamino)cyclopentanecarbonyl-L-phenylalaninal,N-(4-chlorophenylsulfonyl)-L-valyl-L-tryptophanal,N-(4-fluorophenylsulfonyl)-L-valyl-L-tryptophanal,1-(2-naphthalenesulfonylamino)-cyclohexanecarbonyl-L-tryptophanal,N-(2-naphthalenesulfonyl)-L-tert-leucyl-L-tryptophanal,N-(4-fluorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal,N-(2-naphthalenesulfonyl)-L-valyl-L-cyclohexylalaninal,N-(4-chlorophenylsulfonyl)-L-valyl-L-cyclohexylalaninal,N-(4-fluorophenylsulfonyl)-D-valyl-L-leucinal,N-(4-fluorophenylsulfonyl)-L-valyl-D-leucinal,N-(4-fluorophenylsulfonyl)-L-valyl-L-alaninal andN-methylsulfonyl-L-valyl-L-leucinal and the like, which are producedaccording to JP-A-10-147564 (U.S. Pat. No. 6,214,800).

[0038] The lipid usable in the present invention includes a compoundselected from the group consisting of glycerophospholipid(phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol,phosphatidylserine, phosphatidylethanolamine, N-monomethoxypolyethyleneglycol succinyl phosphatidylethanolamine and the like),glyceroglycolipid (digalactosyldiglyceride, galactosyldiglyceride andthe like), sphingophospholipid (sphingomyelin and the like),sphingoglycolipid (cerebroside, ganglioside and the like), sterols(cholesterol, cholesterol hemisuccinate,3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol, ergosterol,lanosterol and the like), synthetic organic compounds having alkyl,alkenyl, alkanoyl or alkenoyl chain having 10-20 carbon atoms (stearicacid, stearylamine, stearic acid hydrazide, stearic acid ester,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride,N-α-trimethylammonioacetyldodecyl-D-glutamate chloride,O-palmitoylpullulan and the like) and the like.

[0039] The above-mentioned lipids (phosphatidylcholine,phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,phosphatidylethanolamine, digalactosyldiglyceride,galactosyldiglyceride, sphingomyelin, cerebroside, ganglioside,N-monomethoxypolyethylene glycol succinyl phosphatidylethanolamine andthe like) each have two saturated or unsaturated fatty acid esterchains, and a lipid wherein the fatty acid (alkanoyl or alkenoyl group)moiety has 10-18 carbon atoms can be used as a constituent component inthe present invention. As the “alkanoyl or alkenoyl group having 10-18carbon atoms”, decylyl, undecylyl, lauroyl, tridecylyl, myristoyl,pentadecylyl, palmitoyl, heptadecylyl, stearoyl, oleoyl group and thelike can be used. Of these, preferred are lauroyl, myristoyl, palmitoyland stearoyl group.

[0040] As the above-mentioned lipid, those generally sold as a reagentand the like can be obtained and used.

[0041] In addition, when a mixture containing a lipid generally havingfatty acid ester chain having various carbon atoms and/or unsaturationdegrees can be obtained, such as egg yolk lecithin, soybean lecithin andthe like, it can be used as it is without separation or purificationinto single components.

[0042] Moreover, the above-mentioned lipid and egg yolk lecithin, aswell as soybean lecithin and the like may be used alone or in a suitablecombination of two or more kinds thereof.

[0043] In the present invention, a lipid having a water-soluble polymerchain can be also used. Examples of such water-soluble polymer chaininclude polyethylene glycol (PEG), polyacrylamide, polysaccharides suchas pullulan, and the like. As a lipid having a water-soluble polymerchain, for example, N-monomethoxypolyethylene glycol succinylphosphatidylethanolamine, O-(C₁₀₋₁₈ alkanoyl or alkenoyl)pullulan,N-(C₁₀₋₁₈ alkanoyl or alkenoyl)polyacrylamide and the like can be used.Preferably, it is N-monomethoxypolyethylene glycol succinyl di(C₁₀₋₁₈alkanoyl or alkenoyl)phosphatidylethanolamine, wherein the PEG moietyhas an average molecular weight of 1000-5000, or O-palmitoylpullulan,most preferably, N-monomethoxypolyethylene glycol succinyl distearoylphosphatidylethanolamine, wherein the PEG moiety has an averagemolecular weight of 2000.

[0044] Using a lipid having such water-soluble polymer, a pharmaceuticalcomposition superior in residence in blood and accumulation property inthe objective tissue can be produced.

[0045] Where necessary, an additive such as α-tocopherol and the like,can be used as a lipid constituting component aiming at an antioxidizingaction and the like.

[0046] The pharmaceutical composition of the present invention means astate where dipeptidyl aldehyde derivative (I) or a pharmaceuticallyacceptable salt thereof, and a lipid form a mixture or a complex.

[0047] The above-mentioned “mixture” may be homogeneous or heterogeneousand refers to the state where dipeptidyl aldehyde derivative (I) or apharmaceutically acceptable salt thereof, and a lipid are admixed,which, upon dispersion in water, permits easy separation of dipeptidylaldehyde derivative (I) or a pharmaceutically acceptable salt thereoffrom the lipid.

[0048] To give a mixture, dipeptidyl aldehyde derivative (I) or apharmaceutically acceptable salt thereof and a lipid are each processedinto fine particles, which are, for example, triturated in a mortar,mechanically mixed in a stirrer, or thoroughly shaken in a plastic bagand the like.

[0049] The above-mentioned “complex” means a state where a bilayermembrane lipid assembly and dipeptidyl aldehyde derivative (I) or apharmaceutically acceptable salt thereof are aggregated (assembly ofmolecules, ions, atoms and the like) by the interaction betweenmolecules such as a cohesive force (e.g., van der Waals force, hydrogenbond, electrostatic interaction and the like), and encompasses a statewhere dipeptidyl aldehyde derivative (I) or a pharmaceuticallyacceptable salt thereof is simply bonded to the surface of a bilayermembrane of a lipid, a state where dipeptidyl aldehyde derivative (I) ora pharmaceutically acceptable salt thereof has partially entered a lipidbilayer membrane, and a state where dipeptidyl aldehyde derivative (I)or a pharmaceutically acceptable salt thereof is completely incorporatedinto a lipid bilayer membrane. The complex is a concept that includes“liposome”. The “liposome” means a closed vesicle consisting of abilayer membrane lipid assembly and an internal aqueous phase, whereindipeptidyl aldehyde derivative (I) or a pharmaceutically acceptable saltthereof generally forms the above-mentioned complex with a membranelipid assembly that constitutes the liposome, or may be enclosed in theaqueous phase in the liposome. As the aqueous phase constituting theinside of a liposome, an aqueous sodium chloride solution, a buffer(phosphate buffer, acetate buffer, sodium dihydrogenphosphate, disodiumhydrogenphosphate and the like), an aqueous solution of monosaccharidesor disaccharides (aqueous solution of glucose, aqueous solution oftrehalose and the like), and the like can be generally used.

[0050] The complex can be prepared by dissolving dipeptidyl aldehydederivative (I) or a pharmaceutically acceptable salt thereof and a lipidin a suitable solvent to give a homogeneous solution, and removing thesolvent. For example, dipeptidyl aldehyde derivative (I) and lecithinare dissolved in an organic solvent such as ether, chloroform, methanol,ethanol and the like to give a homogeneous solution, from which thesolvent is removed and the residue is sufficiently dried.

[0051] The liposome can be obtained by a general production method ofliposomes. For example, phospholipid or glycolipid and the like anddipeptidyl aldehyde derivative (I) are homogenized and a liposome isprepared to encapsulate dipeptidyl aldehyde derivative (I) in theliposome, or a liposome is prepared in advance from phospholipid,glycolipid and the like and dipeptidyl aldehyde derivative (I) may beencapsulated. As the preparation method of liposome, known methods suchas thin membrane method, surfactant removal method, ultrasonicationmethod, ether infusion method, high pressure injection emulsificationmethod, extrusion method and the like are exemplified. According to thethin membrane method, for example, phospholipid is dissolved in anorganic solvent such as ether, chloroform and the like and placed in aglass container such as a round-bottom flask and the like. The organicsolvent is removed by an evaporator or under a nitrogen stream to form alipid thin membrane on a glass surface. By adding an aqueous solutionthereto and applying a mechanical vibration, a liposome suspension canbe obtained. For encapsulation of dipeptidyl aldehyde derivative (I) ina liposome prepared in advance, dipeptidyl aldehyde derivative (I) in asolid state and a liposome suspension are mixed and the mixture isapplied to freeze-thawing or ultrasonication.

[0052] The amount of the lipid to be added to the pharmaceuticalcomposition of the present invention is about 5 parts by weight,preferably about 6 parts by weight, more preferably about 7 parts byweight, for a lower limit, and about 14 parts by weight, preferablyabout 13 parts by weight, more preferably about 12 parts by weight, foran upper limit, per 1 part by weight of dipeptidyl aldehyde derivative(I) or a pharmaceutically acceptable salt thereof. When the amount ofthe lipid to be added is smaller than about 5 parts by weight,absorption property and penetration into tissue of dipeptidyl aldehydederivative (I) or a pharmaceutically acceptable salt thereof are notimproved beyond a certain level, and when it is not less than about 14parts by weight, the dose needs to be increased to ensure sufficientefficacy. When a liposome is prepared and the amount of the lipid to beadded is not less than about 7 parts by weight, dipeptidyl aldehydederivative (I) or a pharmaceutically acceptable salt thereof isencapsulated well in a liposome. When the amount of the lipid to beadded is less than about 7 parts by weight, only a part of thedipeptidyl aldehyde derivative (I) or a pharmaceutically acceptable saltthereof can be encapsulated in a liposome. Therefore, the amount of thelipid to be added to the liposome is preferably about 7-about 14 partsby weight, more preferably about 10-about 12 parts by weight, per 1 partby weight of dipeptidyl aldehyde derivative (I) or a pharmaceuticallyacceptable salt thereof.

[0053] The pharmaceutical composition of the present invention isadministered systemically or topically to a warm-blooded animal (e.g.,human, rat, mouse, rabbit, bovine, swine, dog, cat and the like).

[0054] When the pharmaceutical composition of the present invention isto be produced as a liquid for oral administration, eye drops orinjection, it can be prepared by dispersing a mixture or a complex,preferably a complex, in a solution containing, as appropriate, apharmaceutically acceptable additive such as an isotonicity agent(sodium chloride, potassium chloride, glycerin, mannitol, sorbitol,boric acid, glucose, propylene glycol and the like), a buffer (phosphatebuffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer,Tris buffer, glutamic acid, epsilon aminocaproic acid and the like), apreservative (p-oxybenzoates, benzalkonium chloride, chlorobutanol,benzyl alcohol, sodium dehydroacetate, sodium edetate, boric acid andthe like), a thickener (hydroxyethylcellulose, hydroxypropylcellulose,polyvinyl alcohol, polyethylene glycol and the like), a stabilizer(sodium hydrogensulfite, sodium thiosulfate, sodium edetate, sodiumcitrate, ascorbic acid, dibutylhydroxytoluene and the like), a pHadjusting agent (hydrochloric acid, sodium hydroxide, phosphoric acid,acetic acid and the like) and the like.

[0055] When the pharmaceutical composition of the present invention isto be produced particularly as a liposome preparation for oraladministration, eye drops or injection, it can be prepared by adding apharmaceutically acceptable additive, such as an isotonicity agent(sodium chloride, potassium chloride, glycerin, mannitol, sorbitol,boric acid, glucose, propylene glycol and the like), a buffer (phosphatebuffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer,Tris buffer, glutamic acid, epsilon aminocaproic acid and the like), apreservative (p-oxybenzoates, benzalkonium chloride, chlorobutanol,benzyl alcohol, sodium dehydroacetate, sodium edetate, boric acid andthe like), a thickener (hydroxyethylcellulose, hydroxypropylcellulose,polyvinyl alcohol, polyethylene glycol and the like), a stabilizer(sodium hydrogensulfite, sodium thiosulfate, sodium edetate, sodiumcitrate, ascorbic acid, dibutylhydroxytoluene and the like), pHadjusting agent (hydrochloric acid, sodium hydroxide, phosphoric acid,acetic acid and the like) and the like, to a prepared liposomesuspension.

[0056] While the amount of the additive to be used for preparing thepharmaceutical composition of the present invention into a liquid fororal administration, eye drops or injection, or a liposome preparationfor oral administration, eye drops or injection varies depending on thekind of the additive to be used, use and the like, it may be an amountcorresponding to a concentration capable of achieving the object of theadditive. An isotonicity agent is generally added in a concentration ofabout 0.5-about 5.0 w/v %, to make the osmotic pressure about 229-about343 mOsm. A buffer is added in a concentration of about 0.01-about 2.0w/v %, a thickener is added in a concentration of about 0.01-about 1.0w/v %, a stabilizer is added in a concentration of about 0.001-about 1.0w/v %. A pH adjusting agent is added as appropriate and adjusted togenerally about 3-about 9, preferably about 4-about 8.

[0057] The pharmaceutical composition of the present invention can beprepared into a solid preparation, such as powder, granule, tablet andcapsule and the like. For preparation into granule or tablet, a suitableadditive (lactose, sucrose, glucose, starch, crystalline cellulose andthe like), a sugar alcohol (mannitol, sorbitol and the like), a binder(starch paste solution, hydroxypropylcellulose solution, carmellosesolution, gum arabic solution, gelatin solution, sodium alginatesolution and the like), a disintegrant (starch, carmellose sodium,calcium carbonate and the like), a lubricant (magnesium stearate, talc,stearic acid, calcium stearate and the like) and the like is admixed asappropriate with a mixture or a complex, and according to, for example,general rules for preparations in The Japanese Pharmacopoeia, 14th Ed.,the mixture is prepared into granule or tablet. The granule and tabletmay contain a coloring agent, an aromatic substance, a flavoring agentand the like as necessary. These granule and tablet may be coated with asuitable coating agent (gelatin, sucrose, gum arabic, carnauba wax andthe like), enteric coating agent (e.g., cellulose acetate phthalate,methacrylic acid copolymer, hydroxypropylcellulose phthalate,carboxymethylethylcellulose and the like) and the like. When a capsuleis to be prepared, a mixture or a complex may be filled as it is, or asuitable excipient, such as magnesium stearate, calcium stearate, talc,light silicic anhydride and the like for improving fluidity andlubrication, crystalline cellulose, lactose and the like for compressionfluidity, the above-mentioned disintegrant and the like may be added toa mixture or a complex, and the mixture is uniformly mixed orgranulated, or such granule may be coated with a suitable coating agentand filled, or a suitable excipient and the like are added to a mixtureor a complex and the mixture is encapsulated in a capsule base havingincreased plasticity by adding glycerin, sorbitol and the like to asuitable capsule base (gelatin and the like). Such capsule agents maycontain, where necessary, a coloring agent, a preservative [sulfurdioxide, p-oxybenzoates (methyl p-oxybenzoate, ethyl p-oxybenzoate,propyl p-oxybenzoate and the like)] and the like. The capsule agent maybe processed into a conventional capsule, an enteric-coated capsule, agastric resistant capsule or a controlled release capsule. When anenteric capsule is desired, for example, a mixture coated with theaforementioned enteric coating agent may be filled in a conventionalcapsule or the capsule itself may be coated with an enteric coatingagent, or an enteric polymer may be formed as a capsule base.

[0058] When a solid preparation is produced using liposome as a complex,a suspension of the liposome may be concentrated or lyophilized andsubjected to production as it is, or the above-mentioned suitableadditive and the like are mixed as appropriate to preferably produce asolid preparation. When a liposome is to be lyophilized, addition of acryoprotectant (e.g., monosaccharide, oligosaccharide, sugar alcoholsuch as sorbitol, trehalose and the like) is preferable.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1 shows the results of the intraocular penetration test ofcompound 1 in rabbits in Experimental Example 3.

[0060]FIG. 2 shows the results of the penetration test of compound 1into blood in beagles in Experimental Example 4.

[0061]FIG. 3 shows the results of the penetration test of compound 1into blood in beagles in Experimental Example 5.

BEST MODE FOR EMBODYING THE INVENTION

[0062] The present invention is explained in more detail by referring tothe following Examples and Experimental Examples, which are not to beconstrued as limitative. In the following Examples, ExperimentalExamples and Formulation Examples, compound 1 meansN-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal.

EXAMPLE 1 Compound 1-Containing Complex

[0063] The compound 1 (0.5 g) and egg yolk lecithin (containing about70% of phosphatidylcholine and about 30% of phosphatidylethanolamine;3.5 g) were placed in a 200 mL round-bottom flask and dissolved inchloroform (50 mL). Using a rotary evaporator (RE1-NS, IWAKI),chloroform was removed. The residue was thoroughly dried in a shelf typevacuum dryer to give a complex of compound 1 and egg yolk lecithin.

EXAMPLE 2 Compound 1-Containing Complex

[0064] The compound 1 (1.0 g) and egg yolk lecithin (containing about70% of phosphatidylcholine and about 30% of phosphatidylethanolamine;10.0 g) were placed in a 300 mL round-bottom flask and dissolved inchloroform (100 mL). Using a rotary evaporator (RE1-NS, IWAKI),chloroform was removed. The residue was thoroughly dried in a shelf typevacuum dryer to give a complex of compound 1 and egg yolk lecithin.

EXAMPLE 3 Compound 1-Containing Complex

[0065] The compound 1 (0.5 g) and egg yolk lecithin (containing about70% of phosphatidylcholine and about 30% of phosphatidylethanolamine;2.5 g) were placed in a 200 mL round-bottom flask and dissolved inchloroform (50 mL). Using a rotary evaporator (RE1-NS, IWAKI),chloroform was removed. The residue was thoroughly dried in a shelf typevacuum dryer to give a complex of compound 1 and egg yolk lecithin.

EXAMPLE 4 Compound 1-Containing Complex

[0066] The compound 1 (0.5 g) and phosphatidylcholine dipalmitoyl (3.5g) were placed in a 200 mL round-bottom flask and dissolved inchloroform (50 mL). Using a rotary evaporator (RE1-NS, IWAKI),chloroform was removed. The residue was thoroughly dried in a shelf typevacuum dryer to give a complex of compound 1 and phosphatidylcholinedipalmitoyl. The complex obtained in this Example was subjected tothermal analysis and formation of a complex was confirmed.

EXAMPLE 5 Compound 1-Containing Liposome

[0067] The compound 1 (0.5 g), egg yolk lecithin (containing about 70%of phosphatidylcholine and about 30% of phosphatidylethanolamine; 3.5 g)and cholesterol (1.5 g) were placed in a 200 mL round-bottom flask anddissolved in chloroform (50 mL). Using a rotary evaporator (RE1-NS,IWAKI), chloroform was removed to form a lipid thin membrane on the wallof the flask. A solution (100 mL, pH 7.0) of sodium dihydrogenphosphatedihydrate (0.1 g) and sodium chloride (0.9 g) in sterile purified water(100 mL) was added to this flask, and the mixture was shaken well in avortex mixer. This solution was ultrasonicated in a sonicator (MODELUR-200P, TOMY SEIKO) for 15 min to give a suspension of compound1-containing liposome. The suspension obtained in this Example wasconfirmed to contain compound 1 in a proportion of 0.5 w/v %.

EXAMPLE 6 Compound 1-Containing Liposome

[0068] The compound 1 (1.0 g), egg yolk lecithin (containing about 70%of phosphatidylcholine and about 30% of phosphatidylethanolamine; 7.0 g)and cholesterol (3.0 g) were placed in a 300 mL round-bottom flask anddissolved in chloroform (100 mL). Using a rotary evaporator (RE1-NS,IWAKI), chloroform was removed to form a lipid thin membrane on the wallof the flask. Sterile purified water was added to this flask to make thetotal amount 200 mL. The mixture was shaken well in a vortex mixer. Thissolution was ultrasonicated in a sonicator (MODEL UR-200P, TOMY SEIKO)for 15 min to give a suspension of compound 1-containing liposome. Thesuspension obtained in this Example was confirmed to contain compound 1in a proportion of 0.5 w/v %.

EXAMPLE 7 Compound 1-Containing Liposome

[0069] The compound 1 (0.5 g) and egg yolk lecithin (containing about70% of phosphatidylcholine and about 30% of phosphatidylethanolamine;3.5 g) were placed in a 200 mL round-bottom flask and dissolved inchloroform (50 mL). Using a rotary evaporator (RE1-NS, IWAKI),chloroform was removed to form a lipid thin membrane on the wall of theflask. A 0.1 w/v % aqueous trehalose solution (100 mL) was added to thisflask and the mixture was shaken well in a vortex mixer. This solutionwas ultrasonicated in a sonicator (MODEL UR-200P, TOMY SEIKO) for 10min. The obtained liposome suspension was freeze-dried using a vacuumfreeze dryer (LYPH LOCK6, LABCONCO) to give a freeze dry product ofcompound 1-containing liposome.

EXAMPLE 8 Compound 1-Containing Mixture

[0070] The compound 1 (0.5 g) and egg yolk lecithin (containing about70% of phosphatidylcholine and about 30% of phosphatidylethanolamine;3.5 g) were placed in a mortar and triturated to give a mixture ofcompound 1 and egg yolk lecithin.

EXAMPLE 9 Compound 1-Containing Mixture

[0071] The compound 1 (0.5 g) and phosphatidylcholine dipalmitoyl (3.5g) were placed in a mortar and triturated to give a mixture of compound1 and phosphatidylcholine dipalmitoyl.

EXPERIMENTAL EXAMPLE 1 Stability Test

[0072] (Test Substances)

[0073] Composition of the present invention: A suspension of thecompound 1-containing liposome prepared in Example 5 was used.

[0074] Control liquid: A 0.5 w/v % compound 1-containing suspensionobtained by adding compound 1 to a vehicle (0.9 w/v % sodium chloride,0.1 w/v % sodium dihydrogenphosphate, 0.1 w/v % polysorbate 80, 0.005w/v % benzalkonium chloride; pH 7.0) and ultrasonication thereof usingan ultrasonic cleaner (SUS-103, Shimadzu) at 28 kHz for 5 min into fineparticles was used.

[0075] (Test Method)

[0076] The composition of the present invention and the control liquid(each 5 mL) were respectively filled in a colorless glass ampoule andpreserved in a desktop incubator (CH-M, NAGANO SCIENCE EQUIPMENT MFG.CO., LTD.) set for 60° C. After preservation for 2 weeks and 4 weeks,samples were taken and the residual amount of compound 1 was measuredusing high performance liquid chromatography (HPLC).

[0077] (Test Results)

[0078] The results are shown in Table 1. TABLE 1 Residual amount ofcompound 1 in the composition of the present invention and controlliquid Percent remaining of compound 1 Composition of invention Controlliquid Initial 100.0 100.0 60° C., 2 weeks 91.2 73.2 60° C., 4 weeks76.7 64.1

[0079] The percent remaining of compound 1 in the composition of thepresent invention and the control liquid after preservation at 60° C.for 4 weeks was 76.7% and 64.1%, respectively, and the composition ofthe present invention was superior to the control liquid in thestability of compound 1. From these test results, it was found thatcompound 1 had improved stability when encapsulated in a liposome.

EXP RIMENTAL EXAMPLE 2 Intraocular Penetration Test

[0080] (Test Substances)

[0081] The composition of the present invention (liposome eye drops): Asuspension of the compound 1-containing liposome prepared in Example 5was used.

[0082] Control eye drops: A 0.5 w/v % compound 1-containing suspensionobtained by adding compound 1 to a vehicle (0.9 w/v % sodium chloride,0.1 w/v % sodium dihydrogenphosphate, 0.1 w/v % polysorbate 80, 0.005w/v % benzalkonium chloride; pH 7.0) and ultrasonication thereof usingan ultrasonic cleaner (SUS-103, Shimadzu) at 28 kHz for 5 min into fineparticles was used.

[0083] (Test Method)

[0084] The liposome eye drops and control eye drops were respectivelyinstilled into one eye of a rabbit (male, body weight about 2 kg) by 50μL. After 0.5, 1 and 3 hr of instillation, aqueous humor was taken. Theconcentration of compound 1 in the aqueous humor was measured using HPLC(NANOSPACE SI-1, SHISEIDO) by column switching system.

[0085] (Test Results)

[0086] The results are shown in Table 2. TABLE 2 Penetration test ofliposome eye drops and control eye drops into aqueous humorConcentration (nM)* of compound 1 in aqueous humor Before instillation0.5 hr** 1 hr** 3 hr** liposome eye 0 ± 0 367.1 ± 184.8 6.0 ± 7.2 0 ± 0drops control eye drops 0 ± 0 101.0 ± 56.1   8.6 ± 13.0 4.1 ± 6.4

[0087] In both the liposome eye drops and control eye drops, theconcentration of compound 1 in aqueous humor reached the maximum (Cmax)at 0.5 hr after instillation. The amount of compound 1 in the liposomeeye drops that penetrated into the aqueous humor at Cmax was about 3.6times the amount of that in the control eye drops. From the instant testresults, compound 1 was found to show improved penetration into anaqueous humor by formulating into a liposome.

[0088] The area under the aqueous humor concentration-time curve (AUC)of compound 1 in the aqueous humor is shown in Table 3 for the liposomeeye drops and the control eye drops in 0-3 hr. TABLE 3 AUC of compound 1in aqueous humor AUC_(0→3) (nM · h) liposome eye drops 191.0 control eyedrops 65.3

[0089] The AUC of the liposome eye drops was about 2.9 times higher thanthat of the suspension.

[0090] From the above results, it was found that the composition of thepresent invention was a preparation superior in intraocular penetration.

EXPERIMENTAL EXAMPLE 3 Intraocular Penetration Test

[0091] (Test Substances)

[0092] The composition of the present invention:

[0093] (1) Complex eye drops: A complex (12.0 g) containing compound 1prepared in Example 1 was added to a solution (270 mL, pH 7.0) of sodiumdihydrogenphosphate (0.3 g) and sodium chloride (2.7 g) dissolved insterile purified water, and mixed at 1500 rpm for 30 min using homomixer(ROBOMIX, TOKUSHU KIKA KOGYO CO., LTD.). Sterile purified water wasadded to the mixture to make the total amount 300 mL.

[0094] (2) Liposome eye drops: A suspension of the compound 1-containingliposome prepared in Example 5 was used.

[0095] (3) Mixture eye drops: Sodium dihydrogenphosphate (0.3 g) andsodium chloride (2.7 g) were added to sterile purified water (270 mL)and dissolved. Compound 1 (1.5 g) and egg yolk lecithin (containingphosphatidylcholine (about 70%) and phosphatidylethanolamine (about30%), 10.5 g) were added and the mixture was stirred at 1500 rpm for 30min in a homomixer (ROBOMIX, TOKUSHU KIKA KOGYO CO., LTD.). Sodiumhydroxide was added and the mixture was adjusted to pH 7.0, and sterilepurified water was added to the mixture to make the total amount 300 mL.

[0096] Control eye drops: Polysorbate 80 (0.1 g), sodiumdihydrogenphosphate (0.1 g), benzalkonium chloride (0.005 g) and sodiumchloride (0.9 g) were added to sterile purified water (80 mL) anddissolved. The compound 1 (0.5 g) was added and the mixture wasultrasonicated using an ultrasonic cleaner (SUS-103, Shimadzu) at 28 kHzfor 5 min. Sodium hydroxide was added and the pH was adjusted to 7.0.Sterile purified water was added to the mixture to make the total amount100 mL to give a 0.5 w/v % compound 1-containing suspension, which wasused for the test.

[0097] (Test Method)

[0098] The complex eye drops, liposome eye drops, mixture eye drops andcontrol eye drops were respectively instilled into one eye of a rabbit(male, body weight about 2 kg) by 50 μL. After 0.5, 1 and 3 hr ofinstillation, the rabbit was sacrificed with pentobarbital and aqueoushumor was taken. The concentration of compound 1 in the aqueous humorwas measured using HPLC (NANOSPACE SI-1, SHISEIDO) by column switchingsystem.

[0099] (Test Results)

[0100]FIG. 1 shows the results of concentration of compound 1 in theaqueous humor after administration of the complex eye drops, liposomeeye drops, mixture eye drops and control eye drops. In all samples, theconcentration of compound 1 in aqueous humor reached the maximum at 0.5hr after instillation. The complex eye drops, liposome eye drops andmixture eye drops respectively showed about 3.2 times, about 3.1 timesand about 2.0 times higher concentration than that of the control eyedrops. From the instant test results, composition of the presentinvention was found to be a preparation superior in penetration into thetissue.

EXPERIMENTAL EXAMPLE 4 Penetration into Blood Test

[0101] (Test Substances)

[0102] The composition of the present invention (liposome liquid): Asuspension of the compound 1-containing liposome prepared in Example 6was used.

[0103] Control liquid: A 1.0 w/v % compound 1-containing suspensionobtained by adding compound 1 (1.0 g) to a solution ofcarboxymethylcellulose (0.1 g) in sterile purified water (100 mL) andhomogeneous dispersion was used.

[0104] (Test Method)

[0105] The liposome liquid and control liquid were respectivelyadministered orally using a catheter to a beagle (body weight about 10kg) at a dose of 100 mg/kg of compound 1. Blood was taken at 0.5, 1, 2,4 and 8 hr after the administration. Plasma was separated from the bloodsample and the concentration of compound 1 in plasma was measured usingHPLC (NANOSPACE SI-1, SHISEIDO) by column switching system and taken asthe blood concentration of compound 1.

[0106] (Test Results)

[0107]FIG. 2 shows the measurement results of the blood concentration ofcompound 1 after administration of liposome liquid and control liquid.The liposome liquid showed higher blood concentration than the controlliquid at any measurement times. While both the liposome liquid and thecontrol liquid reached the maximum blood concentration at 2 hr after theadministration, the liposome liquid showed about 3.8 times highermaximum blood concentration than the control liquid. From the testresults, it was found that the composition of the present invention wasa preparation superior in absorption.

EXPERIMENTAL EXAMPLE 5 Penetration into Blood Test

[0108] (Test Substances)

[0109] The composition of the present invention:

[0110] (1) Complex capsule: A complex containing compound 1 prepared inExample 1 was filled in a capsule.

[0111] (2) Liposome liquid: A suspension of the compound 1-containingliposome prepared in Example 5 was used.

[0112] (3) Mixture capsule: Compound 1 (1.0 g) and egg yolk lecithin(containing phosphatidylcholine (about 70%) and phosphatidylethanolamine(about 30%); 7.0 g) were placed in a mortar, triturated and filled in acapsule.

[0113] Control liquid: Compound 1 (0.5 g) was placed in a mortar, 0.5%aqueous carboxymethylcellulose solution (50 mL) was added and trituratedto give a compound 1-containing suspension, which was used for the test.

[0114] (Test method)

[0115] The complex capsule, liposome liquid and mixture capsule wererespectively administered orally to a beagle (body weight about 10 kg)at a dose of 50 mg/kg of compound 1, and the control liquid wasadministered orally to a beagle (body weight about 10 kg) at a dose of100 mg/kg of compound 1. Blood was taken at 0.5, 1, 2, 4 and 8 hr afterthe administration. Plasma was separated from the blood sample and theconcentration of compound 1 in plasma was measured using HPLC (NANOSPACESI-1, SHISEIDO) by column switching system and taken as the bloodconcentration of compound 1.

[0116] (Test Results)

[0117]FIG. 3 shows the measurement results of the blood concentration ofcompound 1 after administration of the complex capsule, liposome liquid,mixture capsule and control liquid. In all administration samples, theblood concentration reached the maximum at 2 hr after administration.The maximum blood concentration was almost the same for the complexcapsule and liposome liquid, and was about 2.4 times and about 2.6times, respectively, higher than that of the control liquid containingcompound 1 in a 2-fold amount relative to that in the complex capsuleand liposome liquid. In the mixture capsule, the amount of penetrationinto blood was slightly lower than that in the control liquid, despitethe fact that the amount of compound 1 was half the amount in thecontrol liquid. From the test results, it was found that the compositionof the present invention was a preparation superior in absorption.Formulation Example 1: eye drops Compound 1 0.2 g phosphatidylcholine1.2 g phosphatidylethanolamine 1.2 g sodium acetate 0.1 g sodiumchloride 0.9 g methyl p-oxybenzoate 0.0026 g propyl p-oxybenzoate 0.0015g sterile purified water total amount (100 mL)

[0118] The compound 1, phosphatidylcholine and phosphatidylethanolaminewere placed in a 100 mL round-bottom flask and dissolved in diethylether. Diethyl ether was removed using a rotary evaporator (RE1-NS,IWAKI) to form a lipid thin membrane on a flask wall surface. To thisflask was added a solution (100 mL, pH 5.0) of sodium acetate, sodiumchloride, methyl p-oxybenzoate and propyl p-oxybenzoate in sterilepurified water and the mixture was shaken well in a vortex mixer. Themixture was ultrasonicated using a sonicator (MODEL UR-200P, TOMY SEIKO)for 10 min to give a compound 1-containing liposome eye drops.Formulation Example 2: injection Compound 1 0.1 g phosphatidylcholine0.7 g cholesterol 0.3 g sodium dihydrogenphosphate 0.1 g sodium chloride0.9 g distilled water for injection total amount (100 mL)

[0119] The compound 1, phosphatidylcholine and cholesterol are placed ina 100 mL round-bottom flask and dissolved in ethanol. Ethanol is removedusing a rotary evaporator (RE1-NS, IWAKI) to form a lipid thin membraneon a flask wall surface. To this flask is added a solution (100 mL, pH7.0) of sodium dihydrogenphosphate and sodium chloride in distilledwater for injection and the mixture is shaken well in a vortex mixer.The mixture is ultrasonicated using a sonicator (MODEL UR-200P, TOMYSEIKO) for 10 min to give a compound 1-containing liposome injection.Formulation Example 3: capsule Compound 1  5 g egg yolk lecithin 50 glactose 45 g

[0120] The compound 1 and egg yolk lecithin (containingphosphatidylcholine (about 70%) and phosphatidylethanolamine (about30%)) are placed in a 100 mL round-bottom flask and dissolved inethanol. Ethanol is removed using a rotary evaporator (RE1-NS, IWAKI) toform a lipid thin membrane on a flask wall surface. To this flask areadded lactose and sterile purified water (100 mL), and the mixture isshaken well in a vortex mixer. The mixture is ultrasonicated using asonicator (MODEL UR-200P, TOMY SEIKO) for 10 min to give a suspension ofcompound 1-containing liposome. The obtained suspension is freeze dried,dispensed and filled in 200 capsules. Formulation Exampl 4: eye dropsCompound 1 0.01 g soybean lecithin 0.1 g sodium dihydrogenphosphate 0.1g sodium chloride 0.9 g benzalkonium chloride 0.005 g sodium hydroxideq.s. sterile purified water total amount (100 mL)

[0121] Sodium dihydrogenphosphate, benzalkonium chloride and sodiumchloride are added to sterile purified water (about 90 mL) anddissolved. Compound 1 and soybean lecithin (containingphosphatidylcholine (about 70%) and phosphatidylethanolamine (about30%)) are added and the mixture is stirred at 3000 rpm for 30 min in ahomomixer (ROBOMIX, TOKUSHU KIKA KOGYO CO., LTD.). Sodium hydroxide isadded and the mixture is adjusted to pH 7.0, and sterile purified waterwas added to the mixture to make the total amount 100 mL. FormulationExample 5: injection Compound 1 0.2 g phosphatidylcholine 0.6 gcholesterol 0.6 g stearylamine 0.4 g sodium chloride 0.9 g sodiumdihydrogenphosphate 0.1 g distilled water for injection total amount(100 mL)

[0122] The compound 1, phosphatidylcholine, cholesterol and stearylamineare placed in a round-bottom flask, and chloroform is added fordissolution. Chloroform is removed by a rotary evaporator (RE1-NS,IWAKI) and a lipid thin membrane is prepared. Thereto is added asolution (100 mL, pH 7.4) of sodium chloride and sodiumdihydrogenphosphate dissolved therein and the mixture is mixed well in avortex mixer.

INDUSTRIAL APPLICABILITY

[0123] The pharmaceutical composition of the present invention showsimproved stability by the coexistence of dipeptidyl aldehyde derivative(I) or a pharmaceutically acceptable salt thereof and a lipid, and showsan absorption-promoting action and improved penetration into tissue.Therefore, it is highly useful as a pharmaceutical preparation for theprophylaxis or treatment of ischemic disease, immune disease,Alzheimer's disease, osteoporosis, diseases due to brain tissueimpairment (e.g., cerebral vasospasm, cerebral thrombosis, cerebralinfarction, cerebral embolism, intracerebral hemorrhage, subarachnoidhemorrhage, hypertensive encephalopathy, transient cerebral ischemicattack, multiinfarct dementia, cerebral arteriosclerosis, Huntington'sdisease and the like), cataract, glaucoma (e.g., open angle glaucoma,low tension glaucoma, closed angle glaucoma and the like),retinochoroidal disorders (e.g., retinal vascular abnormalities such asocclusion of retinal vessels, retinal periphlebitis, Eales' disease,ischemic ophthalmic syndrome, retinal arteriolar macroaneurysm and thelike, retinopathy due to hypertension or renal disease, diabeticretinopathy, retinal pigment epitheliopathy, retinal dystrophy, maculardystrophy, retinochoroidal atrophy, chorioretinopathy, maculardegeneration, macular edema, detachment of the retinal pigmentepithelium, detachment of the retina, degenerative retinoschisis,retinoblastoma, retinal pigment epithelium tumor, optic disc capirallyangioma and the like), eyeball posterior complications due tophotocoagulation (e.g., macular edema, detachment of the retina, opticneuritis, visual field abnormalities, disturbance of light sense, colorvision deficiency and the like) and the like, or as a pharmaceuticalpreparation for the prophylaxis or treatment of angiogenesis, detachmentof the retina and the like.

[0124] While some of the embodiments of the present invention have beendescribed in detail in the above, it will, however, be evident for thoseof ordinary skill in the art that various modifications and changes maybe made to the particular embodiments shown without substantiallydeparting from the novel teaching and advantages of the presentinvention. Such modifications and changes are encompassed in the spiritand scope of the present invention as set forth in the appended claims.

[0125] This application is based on a patent application No. 2000-327677filed in Japan, the contents of which are hereby incorporated byreference.

What is claimed is:
 1. A pharmaceutical composition containing acompound represented by the formula (I)

wherein R¹ is an alkyl group having 1 to 4 carbon atoms or an optionallysubstituted aryl group having 6 to 10 carbon atoms; R² and R³ are thesame or different and each is hydrogen or an alkyl group having 1 to 4carbon atoms, or R² and R³ in combination form a ring having 3 to 7carbon atoms; and R⁴ is a lower alkyl group optionally substituted byaryl, cycloalkyl or aromatic heterocyclic residue, or a pharmaceuticallyacceptable salt thereof and a lipid.
 2. The pharmaceutical compositionof claim 1, wherein R¹ in the formula (I) is phenyl or naphthyl, whichis optionally substituted by fluorine, chlorine or methyl.
 3. Thepharmaceutical composition of claim 1, wherein R¹ in the formula (I) isa group selected from methyl, 4-fluorophenyl, 4-chlorophenyl, p-tolyland 2-naphthyl.
 4. The pharmaceutical composition of claim 1, wherein,in the formula (I), R² is propyl, isopropyl or tert-butyl, and R³ ishydrogen.
 5. The pharmaceutical composition of claim 1, wherein, in theformula (I), R² is isopropyl and R³ is hydrogen.
 6. The pharmaceuticalcomposition of claim 1, wherein, in the formula (I), R² and R³ incombination form cyclopentylidene or cyclohexylidene.
 7. Thepharmaceutical composition of claim 1 wherein, in the formula (I), R⁴ isa group selected from isobutyl, benzyl, cyclohexylmethyl andindol-3-ylmethyl.
 8. The pharmaceutical composition of claim 1, whereinthe compound of the formula (I) isN-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal or a pharmaceuticallyacceptable salt thereof.
 9. The pharmaceutical composition of any ofclaims 1 to 8, wherein a lower limit of the amount of the lipidconstituting the pharmaceutical composition is about 5 parts by weightand an upper limit thereof is about 14 parts by weight, per 1 part byweight of the compound of the formula (I) or a pharmaceuticallyacceptable salt thereof.
 10. The pharmaceutical composition of claim 9,wherein the compound of the formula (I) or a pharmaceutically acceptablesalt thereof and a lipid form a complex.
 11. The pharmaceuticalcomposition of claim 10, wherein the complex is a liposome.
 12. Thepharmaceutical composition of claim 11, wherein the amount of the lipidconstituting the liposome is about 7-about 14 parts by weight, per 1part by weight of the compound of the formula (I) or a pharmaceuticallyacceptable salt thereof.
 13. The pharmaceutical composition of any ofclaims 1 to 12, which is used for the prophylaxis or treatment ofischemic disease, immune disease, Alzheimer's disease, osteoporosis,diseases due to brain tissue impairment, cataract, glaucoma,retinochoroidal disorder, eyeball posterior complication due tophotocoagulation, diseases accompanying angiogenesis and the like. 14.The pharmaceutical composition of any of claims 1 to 13, which is for anoral administration.
 15. The pharmaceutical composition of any of claims1 to 13, which is for eye drops.
 16. The pharmaceutical composition ofany of claims 1 to 13, which is for an injection.
 17. A method forimproving the stability of a compound represented by the formula (I),which comprises bringing a compound represented by the formula (I) or apharmaceutically acceptable salt thereof into contact with a lipid. 18.A method for promoting the absorption of the compound represented by theformula (I), which comprises bringing the compound represented by theformula (I) or a pharmaceutically acceptable salt thereof into contactwith a lipid.
 19. A method for improving penetration of the compoundrepresented by the formula (I) into a tissue, which comprises bringing acompound represented by the formula (I) or the pharmaceuticallyacceptable salt thereof into contact with a lipid.